Dimitrios Coutsinos

Template Usage Is Responsible for the Preferential Acquisition of the K65R Reverse Transcriptase Mutation in Subtype C Variants of Human Immunodeficiency Virus Type 1

ABSTRACT We propose that a nucleotide template-based mechanism facilitates the acquisition of the K65R mutation in subtype C human immunodeficiency virus type 1 (HIV-1). Different patterns of DNA synthesis were observed using DNA templates from viruses of subtype B or C origin. When subtype C reverse transcriptase (RT) was employed to synthesize DNA from subtype C DNA templates, preferential pausing was seen at the nucleotide position responsible for the AAG-to-AGG K65R mutation. This did not occur when the subtype B RT and template were used. Template factors can therefore increase the probability of K65R development in subtype C HIV-1.

Signature Nucleotide Polymorphisms at Positions 64 and 65 in Reverse Transcriptase Favor the Selection of the K65R Resistance Mutation in HIV-1 Subtype C

Recently, we described a novel nucleotide template-based mechanism that may be the basis for the facilitated acquisition of the K65R resistance mutation in subtype C versus subtype B human immunodeficiency virus type 1 (HIV-1). In this article, we evaluated the effects of subtype C-specific silent polymorphisms in cell culture drug-selection experiments using nucleoside and nucleotide reverse-transcriptase inhibitors. The K65R pathway was selected more frequently in a subtype B virus that contained subtype C nucleotide polymorphisms at both positions 64 and 65 than in a wild-type NL4-3 subtype B virus. This is the first demonstration of the significance of silent nucleotide polymorphisms in the development of drug resistance.

Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance

BackgroundWe investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT).MethodsRecombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.ResultsThe efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.ConclusionThe diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

Factors Affecting Template Usage in the Development of K65R Resistance in Subtype C Variants of HIV Type-1

Background: We have shown that the K65R resistance mutation in HIV type-1 (HIV-1) reverse transcriptase (RT) is selected more rapidly in subtype C than subtype B HIV-1 in biochemical, cell culture and clinical studies. Template-usage experiments demonstrated that subtype C nucleotide coding sequences caused RT to preferentially pause, leading to K65R acquisition. This new study now further establishes the basis for differential occurrence of both K65R and thymidine analogue mutations (TAMs) between subtypes. Methods: Gel-based nucleotide extension assays were used to study the homopolymeric sequence surrounding K65. Results: When positive double-stranded DNA synthesis was evaluated from a negative single-stranded DNA template, pausing at the 67 region, which is linked to occurrence of TAMs, was alleviated with both subtype B and C templates at high dCTP concentrations, but this alleviation was more pronounced with the subtype C template. By contrast, pausing at the 65 region on the subtype C but not subtype B template always occurred and was not alleviated at high levels of nucleotide triphosphates or by other means. Furthermore, templates containing repeats of the homopolymeric sequence spanning codons 64–66 of pol showed corresponding pausing repeats at the 65 region with the subtype C template only. Inverted RNA and DNA templates both displayed pausing at position K65 for the subtype C template and a ladder of pausing events culminating at codon 67 for the subtype B templates. Conclusions: These results further establish a mechanistic basis for the exclusion of both K65R and TAMs on single templates as well as the preferential acquisition of K65R in subtype C viruses.

A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

Hepatitis C infection and hepatocellular carcinoma in liver transplantation: a 20-year experience

BACKGROUND Hepatitis C infection (HCV) and hepatocellular carcinoma (HCC), the two main causes of liver transplantation (LT), have reduced survival post-LT. The impact of HCV, HCC and their coexistence on post-LT survival were assessed. METHODOLOGY All 601 LT patients from 1992 to 2011 were reviewed. Those deceased within 30 days (n = 69) and re-transplants (n = 49) were excluded. Recipients were divided into four groups: (a) HCC-/HCV-(n = 252) (b) HCC+/HCV- (n = 58), (c) HCC-/HCV+ (n = 106) and (d) HCC+/HCV+ (n = 67). Demographics, the donor risk index (DRI), Model for End-Stage Liver Disease (MELD) score, survival, complications and tumour characteristics were collected. Statistical analysis included anova, chi-square, Fishers exact tests and Cox and Kaplan-Meier for overall survival. RESULTS Groups were comparable with regards to baseline characteristics, but HCC patients were older. After adjusting for age, MELD, gender and the donor risk index (DRI), survival was lower in the HCC+/HCV+ group (59.5% at 5 yrs) and the hazard ratio (HR) was 1.90 [95% confidence interval (CI),1.24-2.95, P = 0.003] and 1.45 (95% CI, 0.99-2.12, P = 0.054) for HCC-/HCV+. HCC survival was similar to controls (HR 1.18, 95% CI, 0.71-1.93, P = 0.508). HCC+/HCV- patients exceeded the Milan criteria (50% versus 31%, P < 0.04) and had more micro-vascular invasion (37.5% versus 20.6%, P = 0.042). HCC+/HCV+ versus HCC+/HCV- survival remained lower (HR 1.94, 95% CI, 1.06-3.81, P = 0.041) after correcting for tumour characteristics and treatment. CONCLUSION HCV patients had lower survival post-LT. HCC alone had no impact on survival. Patient survival decreased in the HCC+/HCV+ group and this appears to be as a consequence of HCV recurrence.

Kinetics of Inhibition of HIV Type 1 Reverse Transcriptase-Bearing NRTI-associated Mutations by Apricitabine Triphosphate

We wished to investigate the effects of various mutations in HIV-1 reverse transcriptase (RT) on biochemical inhibition by the active form of a novel nucleoside termed apricitabine. Accordingly, we studied the efficiency of chain-termination mediated by apricitabine triphosphate (TP) in cell-free assays that used either recombinant wild-type or mutated RTs. We also performed steady-state-kinetics and primer-unblocking assays. Subtype C RTs were also analysed. The results showed that the K65R mutation in RT caused reductions in the efficiency of chain-termination of apricitabine-TP by increasing its Ki. However, K65R did not affect rates of primer unblocking for apricitabine-TP. No significant differences were found between subtype C and subtype B RTs with regard to any of the parameters studied. Other mutations such as M184V, L74V and K103N had no effect on the efficiency of chain termination by apricitabine-TP. Thus, the mechanism of reduced susceptibility to apricitabine of viruses containing K65R in RT seems to be mediated exclusively through a reduction in binding or incorporation of apricitabine-TP. Unlike some other nucleoside analogues, increased excision of incorporated apricitabine does not seem to be a cause of resistance to apricitabine.

The preferential selection of K65R in HIV-1 subtype C is attenuated by nucleotide polymorphisms at thymidine analogue mutation sites

OBJECTIVES We recently reported the preferential selection of the K65R resistance mutation in subtype C HIV-1 compared with subtype B and showed the underlying mechanism to be dependent on subtype C-specific silent nucleotide polymorphisms, i.e. genomic mutations that change the genotype but not the phenotype. The number of clinical reports demonstrating elevated numbers of K65R nevertheless suggests the existence of factors limiting the increased incidence of K65R mutations. Thus, we investigated the contributions of subtype C-specific silent nucleotide polymorphisms at thymidine analogue mutation (TAM) sites 70, 210 and/or 219 that might reduce the previously described preferential selection of K65R in subtype C HIV-1 associated with subtype C-specific nucleotide polymorphisms at sites 64/65. METHODS Cell culture drug selections were performed with various drugs in MT2 cells. RESULTS The use of nucleoside/nucleotide reverse transcriptase inhibitors [N(t)RTIs] as single drugs or in combination confirmed the more frequent selection of K65R by multiple N(t)RTIs in a subtype B virus that contained the 64/65 nucleotide polymorphisms of subtype C than in a wild-type subtype B virus. This effect was attenuated in the presence of several silent TAM nucleotide polymorphisms, except when stavudine was employed in the selection protocol. CONCLUSIONS These results further demonstrate that stavudine can preferentially select for K65R in subtype C virus and also provide a basis for understanding the importance of silent nucleotide polymorphisms in regard to altered HIV drug resistance profiles.

Antiviral drug development: progress and pitfalls

Conferences on Antiviral Research present a wealth of new information on novel drug development and the 21st annual meeting, held in Montreal between 13–17 April 2008, was no exception. The sessions topics included novel targets for HIV therapy, respiratory and emerging viruses, hepatitis viruses, clinical update on antiviral drugs, retroviruses, and herpes- and poxviruses, among others. In addition, there were a number of themed poster sessions dealing with each of the above areas, as well as novel methods for antiviral analysis, topical microbicides, medicinal chemistry, animal models, veterinary viruses and assorted viral agents. Each of these sessions were well organized and provoked thoughtful discussions. The session on novel targets for HIV therapy featured cutting-edge lectures on interactions between viral proteins and cellular factors. This work has the potential to lead to novel drug discovery as does that on peptide-based approaches to prevent replication of influenza viruses and the developme...